小檗碱调控NLRP3炎症小体对TGF-β1诱导HK-2细胞转分化的影响

Effect of Berberine on TGF-β1 Induced Epithelial Mesenchymal Transition in HK-2 Cells by Regulating NLRP3 Inflammasome

目的:探讨小檗碱(BBR)调控NLRP3炎症小体对转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞(HK-2)转分化的影响。方法:计算机分子对接预测BBR与NLRP3炎症小体的结合情况;采用CCK8法检测不同浓度的BBR对HK-2细胞增殖的影响;将HK-2细胞分为正常组、模型组和小檗碱低、中、高剂量组(10,25,50μmol·L^(-1)),小檗碱预孵18 h后加入TGF-β1刺激48 h,镜下观察各组细胞形态;采用Western blotting法检测E-钙粘蛋白(E-cadherin)、α-平滑肌肌动蛋白(α-SMA)、NOD样受体蛋白3(NLRP3)及半胱氨酸天冬氨酸蛋白酶-1(caspase-1)的表达;caspase-1活性检测试剂盒检测caspase-1的酶活力;采用酶联免疫吸附测定法(ELISA)检测细胞上清液中白细胞介素-1β(IL^(-1)β)的含量。结果:分子对接显示BBR与NLRP3、pro-caspase-1存在结合,可靠性较高。与正常组比较,BBR对HK-2细胞的增殖呈浓度和时间依赖性抑制(P<0.05)。与模型组比较,BBR(10,25,50μmol·L^(-1))可改善HK-2细胞形态,使其趋向椭圆形;上调E-cadherin蛋白和下调α-SMA、NLRP3及caspase-1蛋白的表达(P<0.05);并可降低caspase-1的酶活力、减少IL^(-1)β的分泌(P<0.05)。结论:BBR能改善TGF-β1诱导HK-2细胞的转分化,可能与抑制NLRP3炎症小体活化有关。

Objective:To investigate the effect of berberine(BBR)on transforming growth factor-β1(TGF-β1)induced epithelial mesenchymal transition of human renal tubular epithelial cells(HK-2)by regulating NLRP3 inflammasome.Methods:Computer molecular docking was applied to predict BBR binding to NLRP3 inflammasome.CCK8 assay was used to detect the effect of berberine on the proliferation of HK-2 cells.HK-2 cells were divided into normal group,model group,and low(10μmol·L^(-1)),medium(25μmol·L^(-1))and high dose(50μmol·L^(-1))of BBR groups.TGF-β1 was added for 48-hour stimulation after 18-hour pre-incubation with BBR,and the cell morphology was observed under a microscope.Western blot assay was used to detect the protein expressions of E-cadherin,α-SMA,NLRP3 and caspase-1.Caspase-1 activity detection kit was used to detect caspase-1 activity.ELISA was used to detect the change of IL^(-1)βcontent in the supernatant of HK-2 cells.Results:Molecular docking showed that BBR was combined with NLRP3 and pro-caspase-1 with high reliability.Compared with the normal group,BBR inhibited the proliferation of HK-2 cells in a concentration-time dependent manner(P<0.05).Compared with the model group,BBR at different doses(10,25 and 50μmol·L^(-1))could improve the morphology changes of HK-2 cells,up-regulate the expression of E-cadherin and down-regulate the expressions ofα-SMA,NLRP3 and caspase-1(P<0.05),as well as decrease the enzyme activity of caspase-1 and the secretion of IL^(-1)β(P<0.05).Conclusion:Berberine can alleviate the epithelial mesenchymal transition of HK-2 cells induced by TGF-β1 with the probable mechanism of the inhibition of NLRP3 induced inflammatory activation.